The Role And Mechanism Of GSPE On Renal IR-induced Acute Renal Injury And Chronic Renal Fibrosis

It is increasingly appreciated that acute kidney injury(AKI),which can be cured but result in aberrant incomplete repair,is a major contributor to chronic kidney disease(CKD).In a recent meta-analysis of 13 cohort studies,the pooled incidence rates of CKD and ESRD in patients with AKI were 25.8 and 8.6 per 100 person-years,respectively.AKI can result in persistent tubulointerstitial inflammation,proliferation of fibroblasts and excessive deposition of extracellular matrix,which leading to chronic fibrotic kidney disease.If these reactions can be lessened,it will greatly reduce the ratio of AKI developed to CKD.Grape seed proanthocyanindin extract(GSPE)is a type of polyphenolic bioflavonoid derived from whole grape seeds,with a variety of potent properties such as antioxidant,scavenging oxygen free radicals and anti-inflammatory.Taking into account the commonality of AKI and CKD is inflammation,we administered GSPE in IR-induced acute kidney injury model and chronic kidney fibrosis model.Surprisingly,GSPE can not only alleviate the injury of tubular epithelium and the tubulointerstitial inflammation of AKI,but also mitigate the interstitial fibrosis of CKD.High-mobility group box 1(HMGB1)is a chromatin-binding nuclear molecule thathas potent proinflammatory effects once released to the extracellular.In the kidneys,HMGB1 can be either passively released by necrotic tubular epithelia cells or actively secreted by activate immune cells.After ischemia-reperfusion(I/R),GSPE inhibits the release of HMGB1 and the activation of its effector molecule--p65.Thus attenuate the tubulointerstitial inflammation.In IR-induced chronic kidney fibrosis,GSPE also had the same effect,but not as obvious as those had seen in IR-induced AKI.We considered that if had any other way that GSPE can protect kidney from fibrosis.As the activation and proliferation of fibroblasts also play an important role in chronic kidney fibrosis,we cultured NRK-49F cell lines and stimulated with TGF-?.Interesting,GSPE also can lessen the secretion of extracellular matrix by inhibiting the activation of Smad and JNK signal pathway.These results showed us that,GSPE reduced IR-induced acute renal injury by inhibiting the outbreak of inflammation,but reduced IR-induced chronic renal fibrosis by inhibiting the inflammation of tubulointerstitial and the activation of fibroblasts.Part 1 The role and mechanism of GSPE in Renal IR-Induced Acute kidney injuryObjective To investigate the role and mechanism of GSPE in ischemia reperfusion induced acute kidney injury.Methods C57BL/6 mice were divided into six groups:sham,GSPE-only,bilateral I/R+PBS,bilateral I/R+GSPE(three different doses).Mice were pre-treated with PBS or three different doses of GSPE orally for 5 days before bilateral IR.24h after operation,blood urea nitrogen(BUN)and serum creatinine(Cr)were assayed to assess the renal function.PAS staining,Tunnel assay and the RT-PCR of Kim-1 were used to evaluate the degree of kidney injury.Immunofluorescence and immunohistochemistry were performed to examine the expression and distribution of F4/80,LY6G,MPO and HMGB1.The pro-inflammatory cytokines(IL-6?TNF-??IL-1?)and chemokines(CCL2?CCL3?CCL5?CXCL1?CXCL5?ICAM-1)were detected by RT-PCR.Western blot were used to evaluate the expression of cytoplasmic HMGB1,TLR4 and nuclear P65.Results Compared with mice from the sham and GSPE-only group,mice from I/R+PBS group exhibited significantly increased serum Cr and BUN levels.By contrast,the level of serum Cr and BUN were slightly rising in I/R+GSPE group mice,and GSPE at the concentration of 250mg/kg exhibited the most potent renoprotection.Compared to I/R+PBS group,mice from I/R+GSPE group had lighter tubular necrosis and apoptosis,lower expression of Kim-1,less infiltration of macrophage(F4/80+cells)and neutrophils(LY6G/MPO+cells),less expression of pro-inflammatory cytokines(IL-6?TNF-??IL-1?)and chemokines(CCL2?CCL3?CCL5?CXCL1?CXCL5?ICAM-1).HMGB1 was observed to be predominantly located in the nucleus of renal epithelial cells,little can be seen in cytoplasmic,so as its receptor--TLR4 and effector protein--P65.After reperfusion,HMGB1 transferred to cytoplasmic and release to extracellular,activated its downstream TLR4 and P65.However,the activation of this signaling pathway can be significantly inhibited by GSPE.Conclusions GSPE suppressing the outbreak of inflammation after I/R by inhibiting HMGB1-TLR4-P65 signaling pathway,thus alleviates the acute renal injury.Part 2 GSPE relives chronic renal fibrosis by inhibiting tubulointerstitial inflammationObjective To explore the role of GSPE had played in chronic renal fibrosis,and its potential mechanism of anti-inflammatory.Methods C57BL/6 mice were divided into four groups:sham,GSPE-only,unilateral I/R+PBS,unilateral I/R+GSPE.Mice were administered GSPE for 14 days in succession after they suffered from a unilateral I/R insult.PAS,Masson's and Sirius red staining were selected to evaluate the renal injury and fibrosis.Immunohistochemistry and Western blot were used to detect the expression of PDGFR-??Collagen I?Vimentin??-SMA?cytoplasmic HMGB1,TLR4 and nuclear P65.Immunofluorescence were performed to examine the expression and distribution of F4/80 positive and LY6G positive cells.The diversification of CCL2?IL-6?TNF-??IL-1? were assayed by RT-PCR.Results Masson's and Sirius red staining showed much less severe interstitial fibrosis in GSPE-treated I/R mice than in PBS-treated I/R mice.Western blot and immunohistochemical staining also indicated lower levels of the expression of fibrogenic markers--PDGFR-??Collagen I?Vimentin and ?-SMA in mice from the I/R+GSPE group.So as the change of fibrosis,tubulointerstitial inflammation had the same variety in four groups.In contrast to mice from the I/R+PBS group,mice treated with GSPE can not only limit the infiltration of neutrophils(Ly6G+cells)and macrophages(F4/80+cells),but also attenuate the increase of potent proinflammatory cytokines CCL2?IL-6?TNF-a and IL-1?.Compared to sham and GSPE-only groups,the expression of HMGB1 were elevated in I/R+PBS group.But unlike its distribution in AKI sections,HMGB1 was mainly detected in tubulointerstitial.The change of TLR4 and P65 was the same as HMGB1.However,all of those up-regulation were attenuated by GSPE,but not as apparently as which had seen in AKI models.Conclusions GSPE suppressing the tubulointerstitial inflammation through inhibiting HMGB1-TLR4-P65 signaling pathway,thus alleviates the renal chronic fibrosis.Part 3 GSPE attenuates renal fibrosis by inhibiting the activity of fibroblastObjective To investigate the role and mechanism of GSPE in fibroblasts activate by in vitro experiments.Methods NRK-49F cells were stimulated with lOng/ml TGF-? and intervened with three different doses of GSPE for 48h,Western blot,RT-PCR and cell immunofluorescence were performed to assay the expression of Fibronectin?Collagen I and a-SMA.And collected the protein in the same stimulus at 30min,Western blot was selected to measure the expression of Smad2/3?p-Smad2?p-Smad3?JNK?p-JNK.Results After stimulated with TGF-?,NRK-49F cells had a significantly increased Fibronectin?Collagen I and a-SMA expression.However,the expression of those fibrotic markers were weakened by GSPE,and was positively correlated with the concentration of GPSE.The exchange of p-Smad2,p-Smad3 and p-JNK were as the same as fibrosis,although the total protein Smad2/3 and JNK were unchanged.Conclusions GSPE inhibits the TGF-?induced fibroblasts activate via Smad and JNK signaling pathway suppression.